Metagenomic Study of Microbial Stratification in the Baltic Sea
Introduction The stratification that occurs within the Baltic Sea is as a result of inflows in the Baltic Proper that create strong oxygen and saline gradients from the surface to the bottom, with a layer of stagnant water below the halocline (the vertical zone in the water column with varying salinity/depth that is located beneath the uniform saline surface water layer) (HELCOM, 2009, Wulff & Stigebrandt, 1989). The denser water beneath the halocline is inhibited from vertical mixing, therefore producing hypoxic or anoxic deeper waters (Cederwall & Elmegren, 1990) and resulting in some of the world’s largest marine ‘dead zones’ in the Baltic Proper (Diaz & Rosenberg, 2008). The steep halocline and eutrophication in the Baltic Proper leads to drastic environmental differences and subsequent taxonomic diversity of microbial communities both in the water column (Andersson, Riemann, & Bertilsson, 2010; Koskinen, Hultman, Paulin, Auvinen, & Kankaanpää, 2011; Labrenz, Jost, & Jurgens, 2007; Pinhassi & Hagstrom, 2000; Riemann et al., 2008) and coastal sediment (Edlund, Hårdeman, Jansson, & Sjöling, 2008; Edlund, Soule, Sjöling, & Jansson, 2006). This investigation aimed to establish a degree at which the microbial communities and their associated functional capacities are stratified in the Baltic Proper, specifically at Landsort Deep. The Landsort Deep, as the deepest point in the Baltic Sea, is not strongly affected by inflows leading to sustained stratification with acute anoxia below the halocline (HELCOM, 2003). Methods Sampling Procedure During the spring bloom on April of 2009 at Landsort Deep in the Baltic Sea, Sweden, water and sediment samples were gathered. At a depth of 466 meters, a Kajak sampler was used to collect triplicate sediment cores for further DNA extraction and analysis along with pore water from the sediment top layer using Rhizon Soil Moisture Samplers. Water was also sampled from three unique zones – the surface layer (10-20m), mixed layer (70-80m) and the anoxic zone (400-410m) using Niskin bottles. The samples were then pre-filtered using Sterivex GS filters and further transferred to dry ice prior to DNA sequencing. DNA Extraction and Sequencing The DNA from the sediment top layer was extracted using FastDNA SPIN Kit for Soil and subsequently barcoded using Multiple Identifiers (MIDs) and sequenced with Roche’s 454 GS FLX Titanium pyrosequencing technology. Functional and Taxonomic Annotation Raw metagenomic duplicate sequence reads were aligned to the NCBI non-redundant database using BASTX (Altschul et al., 1997) and further assigned to taxa and SEE (Overbeek et al., 2005) and KEGG (Kanehisa, Goto, Sato, Furumichi, & Tanabe, 2012) functional categories. All downstream analyses only considered bacteria and archeae. Corresponding Analyses Based on data normalized to 100000 reads per dataset, corresponding analyses were done of taxa and functional capacity (SEED hierarchies 1, 2 and 3). The data collected from the environment was further correlated to the correspondence analyses axes using the positions on the axes and Pearson’s correlation coefficient made using Vegan. Global Comparative Metagenomic Analyses The collected and sequenced Landsort Deep data was lastly compared to an array of other marine metagenomes world-wide using the Bray-Curtis distance metric. Data within the range of 0-1 was standardized and subsequent hierarchical clustering was don’t in MeV 4.6 using Kendall’s tau distance metric and average linkage clustering. STAMP (Parks & Beiko, 2010) was then utilized to identify significant taxa and functional abilities for specific metagenomes (or clusters). Results/Discussion Metagenomes that were generated from the four microbial communities of the Baltic Sea (bottom sediment sample and three water column samples) were subjected to correspondence analysis. The analysis revealed significant community stratification, particularly oxygen stratification across the entire study from the surface towards the bottom. Approximately 40-50% of the sequence reads were assigned to taxa and 20% designated a function based on SEED categories. The triplicate sediment samples in the analysis displayed similarities in both taxonomic and functional properties between replicates. The substantial differences in environments across the varying depths were represented by the analysis and the environmental parameters with the largest influence were deduced as dissolved oxygen, salinity and temperature. The microbial community stratification led into the examination of nitrogen and sulfer-dependent functional abilities across the Landsort Deep metagenomic transect. Inorganic nitrogen (most prominently ammonium) accumulates with phosphorous from organic matter mineralization since aerobic nitrification is inhibited. Communities were analyzed for their capacity for denitrification and nitrate/nitrite ammonification and distinct differences were reported. It was noted that a high abundance of these communities in the anoxic water metagenome of Landsort Deep compares to metagenomes from alternative anoxic environments, thus indicating active roles in nitrogen/sulfur transformation in the sampled site. It is known that the importance of nitrogen fixation resembles that of nitrogen removal, and the gene(s) required for this fixation were deduced, allowing further interpretation yielding distinct stratification at the subsystem level across all Landsort Deep communities that increased as a function of depth. Since functional capacity and community taxa were observed to have been influenced by the environment, a metagenomics analysis was performed on both levels with the samples collected from the sediment, soil, and water columns. This analysis aimed to establish similarities amongst microbial communities in similar environments or unique communities that result from the isolation of the environment at Landsort Deep (below the halocline). Strong similarities amongst metagenomes from the Marmara Sea, Californian Tonya Seep and the deepest Landsort Deep microbial communities were observed with an emphasis in genes for regulation and cell signaling, motility and defense mechanisms. Also observed were the close relations between the Landsort Deep surface waters taxonomic profile and three surface water metagenomes from the Western English Channel. These comparative metagenomic results indicate the environment is the main aspect influencing functional capacity and community composition at Landsort Deep. The results from the corresponding analysis performed indicated an array of functional capacities which were further examined in an attempt to determine which underlie stratification. It was observed that, in comparison to all other metagenomes, Landsort Deep metagenomes as a group are largely underregulated in genes involved in biotin biosynthesis but have a large amount of functional capacities such as osmotic regulation and metabolite detoxification of several possible indicators for a stratified environment (Thureborn et al., 2013). References Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). 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